This is an application by a New Investigator to characterize the important molecular and cellular facets of host immunity in high- risk corneal transplantation. High-risk corneal grafts are characterized by their rapid rejection. However, the precise molecular mechanisms that dictate their almost universally poor prognosis remain poorly defined. Therefore, the goal of the proposed studies is to develop new insights into the important immune mechanisms that distinguish the host response in high-risk as compared to low-risk corneal grafts. We hypothesize that the induction of immunity in high-risk corneal transplantation is distinct from that seen in low-risk grafts by virtue of enhanced capacity to activate host T cell responses through both the direct and indirect pathways of sensitization. Moreover, we propose that enhanced immune responses in high-risk corneal grafts are due in part to a distinct profile of chemokines in the eye and draining lymph nodes which induces rapid mobilization of highly immunizing antigen-presenting cells for efficient generation of allodestructive Th1-polarized T cells. We propose to pursue three specific aims: 1. Determine the contribution of the direct and indirect pathways of host allorecognition in low- and high-risk corneal transplantation; 2. Characterize how induction of T cell responses to antigens contained in corneal grafts differs between low- and high-risk transplants; and 3. Determine expression and function of chemokines in mediating graft infiltration and alloimmunity in high-risk corneal transplantation. Our study design relies on: 1. The highly sensitive ELISPOT assay to determine the contribution of the direct and indirect pathways of host sensitization to generation of host T cell responses; 2. Using a transgenic T cell model to time and characterize the induction of immunity to antigens contained in corneal grafts; and 3. Using ribonuclease protection assay, in-situ hybridization, murine knockout models, and neutralizing antibodies in a series of in- vivo and ex-vivo assays to determine the functional role of chemokines in high-risk corneal grafts particularly as they relate to recruitment of antigen-presenting cells in the corneal graft and draining lymph node. The long-term objective of the research program is to utilize the information derived from these projects for developing new prophylactic and treatment strategies to promote graft acceptance, even in the high-risk setting. The overall health relevance of the proposed research is that corneal grafting represents the number one form of tissue transplantation performed in the United States. However, most high-risk patients reject their grafts and the immunosuppressive strategies currently in use are associated with significant toxicity and side-effects.